1. Field of the Invention
The invention in the fields of molecular biology and medicine relates to improved methods and compositions for transfecting cells, particularly dendritic cells and other antigen presenting cells, through the use of cationic liposomes to facilitate transfection with adeno-associated viral (AAV) plasmids. Transfected cells expressing genes of interest, such as tumor-associated or viral antigens, are used for immunization and therapy.
2. Description of the Background Art
Transfection of eukaryotic cells has become an increasingly important technique for the study and development of gene therapy. Advances in gene therapy depend in large part upon the development of delivery systems capable of efficiently introducing DNA into a target cell. A number of methods have been developed for the stable or transient expression of heterologous genes in cultured cell types. These include transduction techniques which use a carrier molecule or virus.
Most gene therapy strategies have relied on transduction by transgene insertion into retroviral or DNA virus vectors. However, adenovirus and other DNA viral vectors can produce infectious sequelae, can be immunogenic after repeated administrations, and can only package a limited amount of insert DNA.
Of the viral vector systems, the recombinant adeno-associated viral (AAV) transduction system has proven to be one of the most efficient vector systems for stably and efficiently carrying genes into a variety of mammalian cell types (Lebkowski, J. S. et al., Mol. Cell. Biol. (1988) 8:3988-3996). It has been well-documented that AAV DNA integrates into cellular DNA as one to several tandem copies joined to cellular DNA through inverted terminal repeats (ITRs) of the viral DNA, and that the physical structure of integrated AAV genomes suggest that viral insertions usually appear as multiple copies with a tandem head to tail orientation via the AAV terminal repeats (Kotin, R. M. et al., Proc. Natl. Acad. Sci. USA (1990) 87:2211-2215). Thus, the AAV terminal repeats (ITRs) are an essential part of the AAV transduction system.
Although recombinant adeno-associated viral (AAV) vectors differ from adenoviral vectors, the transgene DNA size limitation and packaging properties are the same as with any other DNA viral vectors.
AAV is a linear single stranded DNA parvovirus, and requires co-infection by a second unrelated virus in order to achieve productive infection. AAV carries two sets of functional genes: rep genes, which are necessary for viral replication, and structural capsid protein genes (Hermonat, P. L. et al., J. Virol. (1984) 51:329-339). The rep and capsid genes of AAV can be replaced by a desired DNA fragment to generate AAV plasmid DNA. Transcomplementation of rep and capsid genes are required to create a recombinant virus stock. Upon transduction using such virus stock, one recombinant virus uncoats in the nucleus and integrates into the host genome by its molecular ends.
Although extensive progress has been made, transduction techniques suffer from variable efficiency, significant concern about possible recombination with endogenous virus, cellular toxicity and host immune reactions. Thus, there is a need for non-viral DNA transfection procedures.
Liposomes have been used to encapsulate and deliver a variety of materials to cells, including nucleic acids and viral particles (Faller, D. V. et al., J. Virol. (1984) 49:269-272).
Preformed liposomes that contain synthetic cationic lipids have been shown to form stable complexes with polyanionic DNA (Felgner, P. L. et al., Proc. Natl. Acad. Sci. USA (1987) 84:7413-7417). Cationic liposomes, liposomes comprising some cationic lipid, that contain a membrane fusion-promoting lipid dioctadecyldimethyl-ammonium-bromide (DDAB) have efficiently transferred heterologous genes into eukaryotic cells (Rose, J. K. et al., Biotechniques (1991) 10:520-525). Cationic liposomes can mediate high level cellular expression of transgenes, or mRNA, by delivering them into a variety of cultured cell lines (Malone, R. et al., Proc. Natl. Acad. Sci. USA (1989) 86:6077-6081).
Ecotropic and amphotropic packaged retroviral vectors have been shown to infect cultured cells in the presence of cationic liposomes, such as Lipofectin (BRL, Gaithersburg, Md.), and in the absence of specific receptors (Innes, C. L. et al., J. Virol. (1990) 64:957-961).
Even though non-viral techniques have overcome some of the problems of the viral systems, the need still remains for improved transfection efficiency in non-viral systems, increased range of cell types that are transfectable, increased duration of expression in transfected cells, and increased levels of expression following transfection. Some improved efficiency is attained by the use of promoter enhancer elements in the plasmid DNA constructs (Philip, R., et al., J. Biol. Chem. (1993) 268:16087-16090).
Interleukin-2 (IL-2) has been used to treat neoplasms such as metastatic renal cell carcinoma, as one approach to the immune-mediated destruction of human cancer. Although durable complete remissions have been achieved, the overall response rate has been low.
Testing of recombinant IL-2 (rIL-2) (Chiron Corp., Emeryville, Calif.) on cancer patients has revealed dose-limiting toxicity which was dependent upon the route and schedule of IL-2 administration. High dose bolus IL-2 administration was associated with significant toxicity involving nearly every organ system. Moreover, a 4% mortality rate in ECOG 0 performance status patients has been found with high dose IL-2. For an overview of ECOG performance status, see, e.g., Oken, Am. J. Clin. Oncol. (CCT) 5:649-655 (1982), Table 2, at p. 654).
As distinguished from bolus administration, use of lower dose (1-7xc3x97106 Cetus units/M2/day) continuous intravenous infusion of IL-2 has demonstrated clinical efficacy and lowered toxicity, suggesting an improved safety profile in adoptive immunotherapy of advanced cancer (West, W. H. et al., (1987) N. Engl. J. Med. 316:898).
Cell populations which potentially mediate or promote the immune destruction of tumors when combined with IL-2 include lymphokine activated killer (LAK) cells and cytotoxic T lymphocytes (CTL) in particular tumor-infiltrating lymphocytes (TIL). TIL are primarily T lymphocytes found in close apposition to a tumor mass which can be isolated, expanded, and activated in vitro. TIL are of interest in the treatment of neoplasia because of their affinity and presumably their specificity for tumor cells as well as their cytotoxic action. TILs have been reinfused into patients along with exogenous IL-2 (see, e.g., U.S. Pat. No. 5,126,132, Rosenberg, Jun. 30, 1992) which, in some instances, resulted in durable complete remissions of advanced malignancies.
Many attempts have been made to elicit immune responses in subjects that would lead to destruction and removal of unwanted cells, tissues or microorganisms, in particular, tumors or oncogenic viruses such as Epstein-Barr virus (EBV). However, success has been limited. One difficulty is in adequately presenting tumor-associated antigens to the immune system to evoke a cellular immune response. Dendritic cells (xe2x80x9cDCxe2x80x9d; plural is xe2x80x9cDCsxe2x80x9d) are known to be highly potent antigen-presenting cells (xe2x80x9cAPC; plural is (APCsxe2x80x9d). For a review of DCs and their role in immunogenicity, see Steinman, R., Annu. Rev. Immunol. 9:271-296 (1991), which reference is incorporated by reference in its entirety. The present invention employs such cells as a means to present tumor antigens to elicit a specific immune response in vitro or in vivo.
Antigen-specific CTL have been a subject of active investigation for their potential immunotherapeutic utility in the treatment of cancer or virus infection. Tumor-associated antigens (TAA), identified in a number of different types of tumors, including carcinomas can be used for in vitro generation of CTL with lytic activity against cells of the tumor. CEA (carcinoembryonic antigen) is a well-known TAA that is expressed on a majority of colorectal, gastric, pancreatic, non-small cell lung, and breast carcinomas. MART-1 is an example of an antigen associated with melanoma. The present invention targets CEA and melanoma antigens as well as other tumor-associated antigens.
Cationic liposomes are used to facilitate adeno-associated viral (AAV) plasmid transfections of primary and cultured cell types. AAV plasmid DNA complexed with liposomes results in several-fold higher levels of expression of the DNA than do complexes using standard conventional plasmids. In addition, expression lasts for a period of 30 days without any selection. AAV plasmid:liposome complexes induced levels of transgene expression comparable to those obtained by recombinant AAV transduction. High levels of gene expression were observed in freshly isolated CD4+ and CD8+ T cells, TILs and CD34+ stem cells from normal human peripheral blood.
Primary breast, ovarian and lung tumor cells have been transfected using the AAV plasmid DNA:liposome complexes. Transfected tumor cells also expressed the transgene product after lethal irradiation. Transfection efficiency ranged from 10-50% as assessed by J-galactosidase (J-gal) gene expression. The ability to express transgenes in primary tumor cells is utilized to produce tumor vaccines and to generate lymphoid cells that permit highly specific modulations of the cellular immune response in cancer and AIDS, and in gene therapies.
Disclosed herein is a composition for genetic manipulation of host cells which comprises a liposome comprising lipid material and AAV material. The AAV material is preferably a plasmid, such as pMP6-IL2 or pACMV-IL2. The AAV material can comprise an inverted terminal repeat (ITR), or two or more ITRs. Where two ITRs are present in the AAV material, a DNA sequence (or xe2x80x9cgenetic materialxe2x80x9d) of interest can be integrated between the two ITRs. Moreover, a promoter can be integrated between two ITRs. The promoter can be any of a number of promoters which are active in eukaryotic, preferably mammalian, more preferably human cells, such as a CMV immediate-early promoter, a CMV immediate-late promoter, a CMV early promoter, an ADA promoter, or a TK promoter. The composition preferably comprises a DNA sequence of interest, such as a an IL-2 gene or a J-gal gene. The lipid material can comprise a cationic lipid. Also provided are cells transfected by the composition, including antigen-presenting cells, more preferably dendritic cells.
Disclosed herein is a method for introducing a genetic sequence of interest into a host cell. The method comprises steps of providing a composition comprising liposome AAV material end a genetic sequence of interest and contacting the composition with a host cell (which comprises genetic material) whereby the sequence of interest is introduced into the host cell. The host cell can be a CD34+ stem cell, a T cell, such as a CD3+, CD4+, or CD8+ cell, a cell of a tumor cell line such as a bladder cancer, prostate cancer or B lymphoma cell, or an embryonic kidney cell line. Alternatively, the tumor cell may be a primary tumor cell. The step of providing a composition can comprise providing a liposome that comprises cationic lipid. Also included is providing a composition of AAV material that comprises a plasmid, for example, pMP6-IL2 or pACMV-IL2. The method for introducing the genetic sequence of interest into a cell can further comprise a step of integrating the sequence of interest into the genetic material of the host cell.
Disclosed is a method for treating a subject, preferably a human. The treatment method comprises (a) providing a subject with a condition in need of treatment, and (b) providing a composition comprising liposome AAV material and a genetic sequence of interest. The method further comprises a step of contacting the composition with a host cell, whereby the genetic sequence of interest is introduced into the host cell. The contacting step can be in vivo, in which case the host cell is a cell of the subject. Alternatively, the contacting can be ex vivo, in which case the method further comprises a step of delivering the host cell which has been transfected with genetic sequence of interest to the subject. The subject of the present method is preferably one having a condition such as a neoplasm (including a malignant neoplasm), an infection, including HIV infection, an autoimmune condition or a genetic abnormality, such as a missing or defective gene.
The genetic sequence of interest may encode a peptide, an anti-sense oligonucleotide, or RNA. Preferred plasmids to be provided include pMP6-IL2 or pACMV-IL2. The genetic sequence of interest comprises may encode a cytokine, including IL-2 (and may comprise IL-2 genomic DNA, a costimulatory factor, an MHC class I molecule, a tumor-specific or a tumor-associated antigen or the MDR I gene. When the method involves contacting the composition of the invention with a host cell, the host cell may be a neoplastic cell (including a primary tumor cell or a cell of a tumor cell line), a bone marrow hematopoietic cell, a peripheral blood cell or a TIL.
Other preferred plasmids have the pMP6 backbone, as described herein, with any other gene of interest inserted in place of the IL-2 gene of the pMP6-IL2 plasmid
A preferred expression vector comprises a genetic sequence essentially that depicted in FIG. 3 (SEQ ID NO:1). Also preferred is an expression vector which comprises a genetic sequence substantially that of SEQ ID NO:1. In another embodiment, the expression vector comprises a genetic sequence which is SEQ ID NO:1. In a preferred embodiment, the expression vector comprises a genetic sequence essentially that of the genetic sequence of plasmid pMP6, or substantially that of the genetic sequence of plasmid pMP6 or the genetic sequence of plasmid pMP6. An expression vector comprising a genetic sequence essentially that of genetic sequence of plasmid pMP6 preferably further comprises a DNA sequence of interest to be introduced into a cell being transfected, for example a tumor cell or a DC.
Also provided is a cell that is genetically modified with an expression vector comprising a genetic sequence essentially that of SEQ ID NO:1 or with any of the expression vectors listed above. The genetically modified cell can be a cell of any of the categories described above.
The present invention provides a method for producing a protein comprising the steps of providing a composition comprising liposome, AAV material and a genetic sequence of interest. The foregoing composition is contacted with a host cell which comprises genetic material, whereby the genetic sequence of interest is introduced into the host cell. The protein production method further comprises a step of expressing a protein encoded by the genetic sequence of interest. The host cell can be a CD34+ stem cell, a T cell, a cell of a tumor cell line or a primary tumor cell, a TIL, or any CD3+, CD4+, or CD8+ cell. When the cell is from a cell line, preferably a tumor cell line, it may be a bladder cancer cell, a prostate cancer cell, a B lymphoma, or a cell of an embryonic kidney cell line.
The composition provided in the protein production method, as described above, may comprises cationic lipid and AAV material. The AAV material preferably comprises a plasmid, such as pMP6-IL2 or pACMV-IL2.
The above method for producing a protein can comprise a further step of integrating the genetic material of interest into the genetic material of the host cell. The step of expressing a protein can comprise expressing a lymphokine, such as IL-2, a lymphokine analog, the product of the MDR-I gene or a marker or reporter product such as J-gal or chloramphenicol-acetyl-transferase (CAT)
Another objective of the present invention is to provide methods for generating tumor antigen-specific CTL for use in adoptive immunotherapy. An approach taken by the present inventors involved the use of DC to express and present the desired tumor antigen either by direct loading of the DC with the antigen or by transfection of DC with genetic constructs which will result in expression of the antigen. Such transfections are preferably accomplished using the methods and compositions described herein, namely AAV plasmid DNA (which includes DNA encoding the TAA) complexed with cationic liposomes. The AAV plasmid/cationic liposome methods and compositions are also used to express other antigens on DC, for example, viral antigens, preferably HIV antigens which serve as the target of an effective antiviral T cell or antibody response.
The invention is directed, in one aspect, to the expression of nonendogenous peptides or proteins, in particular TAA or a viral antigen, in or on the surface of DC. The invention is further directed to the use of DC as APCs to generate CTL capable of killing tumor cells or virus-infected cells bearing the antigen. The antigens are provided to the DC either by pulsing the cells with the desired peptide or by transfecting the cells with a vector capable of expressing the desired antigen whereby the DC can appropriately present the peptide on its surface.
Given applicants"" discovery that antigen presentation by C cells to CTL provokes a very effective response by CTL, it is believed that a variety of other methods for providing a given protein or peptide antigen to a DC cell can be used to generate CTL capable of killing tumor cells or virus infected cells which are bearing the antigen. The DC are preferably used to stimulate a potent reactive lymphocyte population, preferably CTL, in culture, and such lymphocytes are then administered to the subject to effect treatment of a condition such as a tumor or virus infection. Alternatively or additionally, as a direct form of immunotherapy, the DC expressing the desired antigen in immunogenic form are administered to a subject and used to elicit a CTL response or other protective immune response in vivo.
The present invention provides a non-immortalized DC transfected by a vector including a DNA sequence not native to the DC. Also provided is a DC or other APC transfected by a composition comprising a liposome (which comprises lipid material) and AAV material. The AAV material preferably comprises a plasmid which preferably includes a DNA sequence of interest encoding one or more of a tumor-specific antigen, a tumor-associated antigen, a microbial antigen, a cytokine, a cellular receptor, a reporter molecule or a selectable marker. A preferred plasmid is pMP6 in which is inserted the DNA sequence of interest.
This invention is further directed to a method for introducing a DNA sequence of interest into a DC or other APC comprising the steps of:
(a) providing a composition comprising liposome, preferably cationic lipid, AAV material, preferably a plasmid, and a DNA sequence of interest; and
(b) contacting the composition of step (a) with the cell, which cell comprises genetic material, such that the DNA sequence of interest is introduced into the cell. A preferred plasmid is pMP6 in which is inserted the DNA sequence of interest.
In the above method, the DNA sequence of interest preferably encodes one or more of a tumor-specific or tumor-associated antigen, a microbial antigen, a cytokine, a cellular receptor, a reporter molecule or a selectable marker. The tumor-specific or tumor-associated antigen may be carcinoembryonic antigen, a breast tumor antigen, a colorectal tumor antigen, a gastric tumor antigen, a pancreatic tumor antigen, a lung tumor antigen, an ovarian tumor antigen, a bladder tumor antigen, a prostate tumor antigen, a melanoma antigen, a leukemia antigen or a lymphoma antigen. The microbial antigen may be any viral or bacterial antigen. Preferably viral antigens are human retrovirus or human DNA virus antigens, preferably Epstein-Barr viral antigen or an HIV-1 or HIV-2 antigen. The cytokine is preferably IL-2. The receptor may be nerve growth factor receptor. A preferred reporter molecule is bacterial chloramphenicol acetyl transferase.
In the foregoing method, the DNA sequence of interest may or may not integrate into the genetic material of the transfected cell.
The present invention provides a method for treating a subject having a disease or condition which is treatable by stimulating an immune response to a selected antigen in the subject, which method comprises the steps of:
(a) contacting DCs or other APCs of the subject in vivo with a composition comprising liposome, adeno-associated virus material and a DNA sequence of interest which encodes the selected antigen such that the DNA sequence is introduced into the cells; and
(b) allowing the antigen encoded by the DNA sequence to be expressed and to stimulate the immune response of the subject,
thereby treating the subject.
In a related embodiment, the method comprises steps of:
(a) contacting dendritic cells or other antigen-presenting cells, which cells are autologous or allogeneic to the subject, ex vivo with a composition comprising liposome, adeno-associated virus material and a DNA sequence of interest which encodes the selected antigen such that the DNA sequence is introduced into the cells;
(b) allowing the antigen encoded by the DNA sequence to be expressed in the cells; and
(c) delivering the cells expressing the antigen to the subject to stimulate the immune response, thereby treating the subject.
In yet another related embodiment, the method comprises the steps of:
(a) contacting dendritic cells or other antigen-presenting cells, which cells are autologous or allogeneic to the subject, ex vivo with a composition comprising liposome, adeno-associated virus material and a DNA sequence of interest which encodes the selected antigen such that the DNA sequence is introduced into the cells;
(b) allowing the antigen encoded by the DNA sequence to be expressed in the cells;
(c) activating lymphocytes ex vivo by contacting them with the cells expressing the antigen such that the lymphocytes become cytotoxic or otherwise specifically immunoreactive to host cells bearing the antigen; and
(d) delivering the activated lymphocytes to the subject to mediate the immune response in the subject, thereby treating the subject.
In the foregoing treatment methods, the condition or disease to be treated may be neoplasia or an infection. Examples of neoplasia which are treated by these methods include breast cancer, colorectal cancer, gastric cancer, pancreatic cancer, lung cancer, ovarian cancer, bladder cancer, prostate cancer, melanoma, leukemia and lymphoma.
Infections which may be treated by these methods include infection with a human retrovirus, preferably HIV-1 or HIV-2, HTLV-1, HTLV-2 and the like, or a human DNA virus, preferably Epstein-Barr virus or other herpesviruses.
In the foregoing treatment methods, the DNA sequence of interest preferably encodes one or more of a tumor-specific antigen, a tumor-associated antigen or a microbial antigen. Tumor-associated or tumor-specific antigens include carcinoembryonic antigen, a breast tumor antigen, a colorectal tumor antigen, a gastric tumor antigen, a pancreatic tumor antigen, a lung tumor antigen, an ovarian tumor antigen, a bladder tumor antigen, a prostate tumor antigen, a melanoma antigen, a leukemia antigen or a lymphoma antigen. Microbial antigens include bacterial or viral antigens, preferably an HIV antigen, for example, an epitope of a protein encoded by the HIV gag, pol, env or nef gene.
In the foregoing treatment methods, the DNA sequence of interest may further encode a cytokine, a costimulatory factor or an antigen of an MHC class I molecule.
This invention is also directed to a method for producing a protein in a dendritic cell comprising:
(a) introducing a DNA sequence encoding the protein into a dendritic cell; and
(b) allowing the DNA sequence to be expressed,
thereby producing the protein. The introducing is preferably performed by transfecting the cell with a composition comprising liposome, adeno-associated virus material and the DNA sequence, as described herein.
Another aspect of the present invention is the provision of a method for eliciting an immune response to a tumor-associated antigen comprising providing a modified dendritic cell bearing a selected tumor-associated antigen and contacting a cytolytic T cell with said dendritic cell bearing said tumor-associated antigen. The contacting can occur in vivo or in vitro. The term xe2x80x9cmodifiedxe2x80x9d refers to a dendritic cell that has been changed so that it bears a tumor-associated antigen that has been selected based on the nature of the tumor in a patient to be treated.